Project Summary CRISPR offers the promise of total control over genes in model organisms, such as the nematode C. elegans. However, any individual edit takes on the order of 6 weeks from beginning to end. To edit many genes is just not practical. The goals of this project are to make CRISPR genome modifications simple and fast, and to increase the throughput. We propose a series of multiplexed genome engineering methods that will accelerate gene tagging in C. elegans by one to two orders of magnitude. First, we propose to develop cassette exchange methods that will allow geneticists to alter one gene with many tags or knockout strategies. Second, we propose to develop a multiplexed CRISPR strategy that will allow groups to modify many genes within a single editing experiment. Third, we will develop reagent libraries capable of modifying all genes in the genome for distribution to the community. Aim 1. One gene: recombinase-mediated cassette exchange. We will develop a cassette exchange method for rapidly integrating transgenes at a defined locus in the genome. Aim 2. Many genes: multiplex CRISPR strain. To enable efficient and easy editing of many genes at once, we will create methods and reagents for performing many CRISPR edits in parallel. Aim 3. All genes: tagged-gene collection. We will create a cost-effective pooled workflow for building genome editing reagents, then use these reagents to endogenously tag 1000 neuronally expressed genes with GFP. C. elegans shares most of the genes mutated in human genetic diseases, making it a major model for studying the function of these genes in a simple, compact, and rapidly developing animal. In the future, the genome engineering pipelines developed here could be used to tag every protein-coding gene in the C. elegans genome with a variety of functionally distinct tags. Such a strain collection would be a boon for cell biologists and geneticists, enabling new inroads in studying how organisms work and how to fix what goes awry in disease.